The Ve-resistance locus, a plant signaling intercept.

MAIN CONCLUSION: The Ve-resistance locus in tomato and potato affects both stress/defense and growth, consistent with a signaling intercept and a competitive regulatory mechanism. Acting in an antagonistic fashion, the two genes comprising the tomato Ve-resistance locus have been shown to influence both the defense/stress cascade, which causes wilt symptoms, and plant growth (Nazar et al. in Planta 247:1339-1350, 2018c); in contrast, both have been reported to elevate wilt resistance in potato or Arabidopsis. In a further examination of this influence in potato transformed with the Ve1 gene, effects are again demonstrated with respect to both disease resistance and crop productivity consistent with the Ve locus being a signaling intercept and the antagonistic effects, previously observed in tomato. The results support a competitive model in which the tomato Ve1 and Ve2 proteins act to reduce the detrimental effects of the defense/stress cascade and energy transfers to the developing potato tubers.

Wounding induces tomato Ve1 R-gene expression.

MAIN CONCLUSION: In tomato, Ve1 gene expression is induced specifically by physical damage or plant wounding, resulting in a defense/stress cascade that mimics responses during Verticillium colonization and wilt. In tomato, Verticillium resistance is determined by the Ve gene locus, which encodes two leucine-rich repeat-receptor-like proteins (Ve1, Ve2); the Ve1 gene is induced differentially while Ve2 is constitutively expressed throughout disease development. These profiles have been observed even during compatible Verticillium interactions, colonization by some bacterial pathogens, and growth of transgenic tomato plants expressing the fungal Ave1 effector, suggesting broader roles in disease and/or stress. Here, we have examined further Ve gene expression in resistant and susceptible plants under abiotic stress, including a water deficit, salinity and physical damage. Using both quantitative RT-PCR and label-free LC-MS methods, changes have been evaluated at both the mRNA and protein levels. The results indicate that Ve1 gene expression responds specifically to physical damage or plant wounding, resulting in a defense/stress cascade that resembles observations during Verticillium colonization. In addition, the elimination or reduction of Ve1 or Ve2 gene function also result in proteomic responses that occur with wilt pathogen and continue to be consistent with an antagonistic relationship between the two genes. Mutational analyses also indicate the plant wounding hormone, systemin, is not required, while jasmonic acid again appears to play a direct role in induction of the Ve1 gene.

Antagonistic function of the Ve R-genes in tomato.

Key message In Verticillium wilt, gene silencing indicates that tomato Ve2-gene expression can have a dramatic effect on many defense/stress protein levels while Ve1-gene induction modulates these effects in a negative fashion. In tomato, Verticillium resistance is dependent on the Ve R-gene locus, which encodes two leucine-rich repeat receptor-like proteins, Ve1 and Ve2. During fungal wilt, Ve1 protein is sharply induced while Ve2 appears expressed constitutively throughout disease development; the disease resistance function usually is attributed to the Ve1 receptor alone. To study Ve2 function, levels of Ve2 mRNA were suppressed using RNAi in both susceptible and resistant Craigella tomato near-isolines and protein changes were evaluated at both the mRNA and protein levels. The results indicate that Ve2-gene expression can have dramatic effects on many defense/stress protein levels while the presence of intact Ve1 protein minimizes these effects in a negative fashion. The data suggest an antagonistic relationship between the Ve proteins in which Ve1 modulates the induction of defense/stress proteins by Ve2.

Defence cascade in Verticillium-infected grafted tomato.

In tomato the Ve1-gene provides resistance to the vascular pathogen, Verticillium dahliae, race 1; ve1 plants are susceptible. Reciprocal grafts of resistant and susceptible tomato near-isolines were used to examine proteomic changes and, in particular, the effect of the Ve1-gene on the defence/stress protein cascade induced during Verticillium wilt. Based on label-free LC-MS, the results indicate that this defence response is cell-specific, correlates with overall fungal colonization and is mitigated by Ve1 function. The influence of the Ve1-gene in resistant tissues, however, is not actually transferred to susceptible tissues in the grafted plant.

Tomato Ve resistance locus; defense or growth.

MAIN CONCLUSION: Verticillium colonization does induce a cascade of defense/stress proteins but the Ve1 gene also promotes enhanced root growth, which appears to allow the plant to outgrow the pathogen and avoid symptoms associated with an exaggerated defense response. In tomato, the Ve1 gene provides resistance to the vascular pathogen, Verticillium dahliae, race 1; ve1 plants are susceptible. However, the physiological basis of the resistance is unknown. While developing alternative lines of mutant Ve1 gene transformants to address this question a striking difference was observed in transformation frequency resulting from the inefficient rooting of plantlets from ve1 callus relative to Ve1 callus. Subsequent experiments with resistant and susceptible near-isolines of the cultivar Craigella, as well as Ve1 transformants, showed that in both artificial medium and soil, root growth was significantly enhanced in the resistant cultivar. Parallel studies of Verticillium colonization indicated a significantly lower overall concentration in the resistant plant characteristic of the resistant phenotype, but an almost equal total fungal biomass in both resistant and susceptible roots. Proteomic analyses of the roots of Verticillium-infected plants revealed elevated levels of defense/stress proteins, which correlated with the fungal concentration rather than resistance. Hormone analyses demonstrated a higher cis-ABA level in the resistant isoline consistent with enhanced root growth. Taken together these studies indicate a similar fungal biomass in the roots of both isolines where the Ve1 gene also promotes root production. In the case of the Craigella/Vd1 pathosystem, this appears to allow the host to resist better by outgrowing the pathogen with less wilt rather than reliance only on partial immunity.

Biotic factors that induce the tomato Ve1 R-gene.

In tomato, Verticillium resistance is determined by the Ve gene locus encoding two leucine-rich repeat-receptor-like proteins (Ve1, Ve2). The resistance function usually is attributed to Ve1 alone, with two known alleles: Ve1, encoding a resistance protein, and ve1, with a premature stop codon encoding a truncated product. We have examined further Ve-gene expression in resistant and susceptible near-isolines of Verticillium-infected Craigella tomatoes, using both quantitative RT-PCR and an alternative RFLP assay. Ve1 is induced differentially in resistant and susceptible plants, while Ve2 is constitutively expressed throughout disease development. Contrary to their putative role in Verticillium resistance, these profiles were observed even with compatible Verticillium interactions, some bacterial pathogens, and transgenic tomato plants expressing the fungal Ave1 effector. This suggests broader roles in disease and/or stress. To determine the contribution of plant hormones, abscisic acid, methyl jasmonate, naphthaleneacetic acid or salicylic acid were infused independently via the tomato root and effects on Ve1 induction were confirmed using biosynthesis mutants. While all the hormones modulated Ve1-gene induction, abscisic acid and salicylic acid were not required while jasmonic acid appears to play a more direct role.

Verticillium Ave1 effector induces tomato defense gene expression independent of Ve1 protein.

Verticillium resistance is thought to be mediated by Ve1 protein, which presumably follows a "gene-for-gene" relationship with the V. dahliae Ave1 effector. Because in planta analyses of Ave1 have relied so far on transient expression of the gene in tobacco, this study investigated gene function using stably expressing 35S:Ave1 transgenic tomato. Transgenic Ave1 expression was shown to induce various defense genes including those coding for PR-1 (P6), PR-2 (ßbeta-1,3-glucanase) and peroxidases (anionic peroxidase 2, Cevi16 peroxidase). Since a Ve1- tomato cultivar served as germplasm, these results indicate that Ave1 induces these defense genes independently of Ve1.

Arsenal of elevated defense proteins fails to protect tomato against Verticillium dahliae.

Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.

DNA chip analysis in diverse organisms with unsequenced genomes.

Whether for basic research or biotechnology, DNA microarrays have become indispensable tools for studying the transcriptome. Normally, analyses begin with a set of known cDNA sequences to prepare microarray chips specific for a target organism with an extensively sequenced and annotated genome. For many organisms, however, genome programs are not complete or have not been initiated. The present study demonstrates that, whether using homologous or heterologous arrays, the chances of seeing interesting differences are similar. When a specific DNA microarray is not available, the results indicate that a reverse approach based on a heterologous array can be used to probe for interesting differences in gene expression. This may be sufficient in many studies but, if necessary, the genes exhibiting the most significant changes subsequently could be identified by traditional molecular approaches. Such a reverse strategy can provide a convenient and inexpensive approach to probe for significant genetic changes in many diverse studies, to monitor or mine critical biological information for basic or applied research, long before complete sequence data are available.

Endophyte-induced Verticillium protection in tomato is range-restricted.

Endophytes, bacterial, fungal or viral, colonize plants often without causing visible symptoms. More important, they may benefit host plants in many ways, most notably by preventing diseases caused by normally virulent pathogens. Previous studies have shown that an isolate of V. dahliae from eggplant, Dvd-E6, can colonize tomato endophytically, producing taller and more robust tomato plants while providing protection against a virulent V. dahliae, race 1 (Vd1) isolate. Expression analyses suggest this requires interplay between Dvd-E6 and the plant that involves resistance and defense genes. To examine the possibility of a broader effect, dual interactions have been further examined with a more distantly related pathogen, Verticillium albo-atrum (Vaa). The results indicate Dvd-E6 colonization selectively modifies the expression of specific tomato genes to be detrimental to Vd1 but not Vaa, providing evidence that Verticillium-induced protection is range-restricted.

Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1.

Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant diseases worldwide. The Verticillium genus includes vascular wilt pathogens with a wide host range. Although V. longisporum infects various hosts belonging to the Cruciferaceae, V. dahliae and V. albo-atrum cause vascular wilt diseases in over 200 dicotyledonous species, including economically important crops. A locus responsible for resistance against race 1 strains of V. dahliae and V. albo-atrum has been cloned from tomato (Solanum lycopersicum) only. This locus, known as Ve, comprises two closely linked inversely oriented genes, Ve1 and Ve2, that encode cell surface receptor proteins of the extracellular leucine-rich repeat receptor-like protein class of disease resistance proteins. Here, we show that Ve1, but not Ve2, provides resistance in tomato against race 1 strains of V. dahliae and V. albo-atrum and not against race 2 strains. Using virus-induced gene silencing in tomato, the signaling cascade downstream of Ve1 is shown to require both EDS1 and NDR1. In addition, NRC1, ACIF, MEK2, and SERK3/BAK1 also act as positive regulators of Ve1 in tomato. In conclusion, Ve1-mediated resistance signaling only partially overlaps with signaling mediated by Cf proteins, type members of the receptor-like protein class of resistance proteins.

Plant-endophyte interplay protects tomato against a virulent Verticillium.

Endophytes, bacterial, fungal or viral, colonize plants often without causing visible symptoms. More important, they may benefit host plants in many ways, most notably by preventing diseases caused by normally virulent pathogens. Craigella tomatoes (Lycopersicon esculentum Mill.) can be infected with Verticillium dahliae Kleb., either race 1 (Vd1) or a non-host isolate Dvd-E6 resulting in susceptibility or tolerance, respectively. The present study sought to determine whether Dvd-E6 is endophytic and can protect tomato against Vd1. The total amount of Verticillium in stems and roots was determined by quantitative PCR; the relative amounts of Vd1 and Dvd-E6 were assessed by restriction fragment polymorphism. When Dvd-E6 infects before or together with Vd1, Vd1 is excluded almost completely from the root but, when Vd1 infects first, Dvd-E6 can compete on an equal basis. Previous studies suggested that Dvd-E6 suppresses symptom-related genes, raising the possibility that Dvd-E6 simultaneously induces tolerance to Vd1. This does not seem to be entirely the case since the minimal symptoms following Vd1 infection of Dvd-E6 tolerant Craigella result, at least in part, from restricted Vd1 colonization. Furthermore, when Vd1 and Dvd-E6 are cultured on PDA plates alone or together, the growth rates are similar and neither is inhibitory to the other. Dvd-E6 does not outgrow or inhibit Vd1, in vitro. The protective effect apparently requires interplay between Dvd-E6 and the plant. Expression analyses of tomato genes involved in resistance and defence support this interpretation.

Gene suppression in a tolerant tomato-vascular pathogen interaction.

A plant can respond to the threat of a pathogen through resistance defenses or through tolerance. Resistance has been widely studied in many host pathogen systems but little is known about genetic changes which underlie a tolerant interaction. A recently developed model system for a tolerant tomato (Lycopersicon esculentum Mill) interaction with a fungal wilt pathogen, Verticillium dahliae Kleb, is examined with respect to changes in gene expression and compared to a susceptible infection. The results indicate that genetic changes can be dramatically different and some genes that are strongly elevated in the susceptible interaction are actually down-regulated in tolerance. Similar levels of fungal DNA and an up-regulation of many pathogenesis related genes indicate that in both types of interaction the presence of fungus is clearly recognized by the plant but other changes correlate with the absence of symptoms in the tolerant interaction. For example, a gene encoding a known 14-3-3 regulatory protein and a number of genes normally affected by this protein are down-regulated. Furthermore, genes which may contribute to foliar necrosis and cell death in the susceptible interaction also appear to be suppressed in the tolerant interaction, raising the possibility that the wilt symptoms, chlorosis and necrosis which are observed in the susceptible interaction, are actually programmed to further limit the growth of the fungal pathogen, and protect the general tomato population.